ESC/iPSC-derive somatic cells may be ideal for treating disorders caused by cellular deficiency or dysfunction. To form a lineage-specific cell population, ESCs/iPSCs undergo a multi-step process that recapitulates embryonic development. ESC/iPSC differentiation protocols are hampered by the limitation of our understanding in development. Zebrafish embryos are fertilized and developed externally, a feature facilitates the observation and manipulation of embryonic development. To explore the zebrafish as a system to study cell lineage determination, in this thesis, I 1) identified an ortholog of the key pluripotency regulator Nanog in zebrafish and examined its role in early cell fate determination; 2) developed a high-throughput image-based chemical screening system in zebrafish blastomere cell culture that is very similar to, but much faster than, ESC/iPSC differentiation screens. Specifically, in an effort to examine the role of Nanog in vivo, I identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. I examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. The study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction. In the second part of the thesis, I developed a system that allows high-throughput image-based chemical screening using cultured zebrafish blastomere cells. To demonstrate its potential, this system is utilized to study skeletal muscle development. I screened 2,400 chemicals, finding 11 chemicals that block mature muscle cell differentiation and 17 chemicals that block skeletal muscle progenitor formation. The subsequent studies of these hits illustrate an RTK-PI3K-mTOR- GSK3 signaling cascade that is critical for skeletal muscle development. Preliminary data in mouse Skeletal Muscle Precursors (SMPs) suggest the pathway is conserved in murine adult muscle stem cells. This system, which can be modified for any cell lineage, promises to enhance our understanding of fundamental biology and to identify chemicals for cell-based therapies for many diseases.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10288405 |
| Date | January 2012 |
| Creators | Xu, Cong |
| Contributors | Zon, Leonard Ira |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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